exoRNeasy Serum Plasma Kits
For efficient purification of RNA from exosomes and other extracellular vesicles out of serum or plasma samples
exoRNeasy Serum/Plasma Kits use membrane affinity spin columns to efficiently isolate RNA from exosomes and other extracellular vesicles extracted from serum or plasma. The midi column enables efficient processing of smaller sample volumes (1 ml or less), while the maxi column format allows the use of large sample volumes, up to 4 ml, to detect low-abundance RNAs with high confidence. The results are fast, consistent, and highly suited to sensitive downstream applications. The exoRNeasy Serum/Plasma Kits can be automated on the QIAcube.
exoRNeasy Serum Plasma Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
Performance
The protocol for exoRNeasy purification of exosomes and other extracellular vesicles is not only faster than ultracentrifugation, but also yields a cleaner preparation. Scanning electron microscopy shows that while both techniques deliver intact vesicles of the expected size, the preparation from ultracentrifugation also contains many smaller structures that do not match the expected size or shape for EVs (see figure Intact vesicles are eluted from the exoEasy membrane with higher purity compared with ultracentrifugation). Moreover, both exoRNeasy and the traditional ultracentrifugation method deliver similar sizes and yields of large and small RNAs, as measured by Bioanalyzer analysis (see figure exoRNeasy isolates small and large RNAs from extracellular vesicles in plasma). Finally, exoRNeasy captures all cell-free mRNAs in plasma, as well as EV-associated miRNAs, as confirmed by RT-qPCR experiments on the RNAs purified with the exoRNeasy protocol and the flow-through from the exoEasy column (see figure exoRNeasy captures all mRNA and vesicle-specific miRNAs in plasma).
Principle
The exoRNeasy Serum/Plasma Kits improve upon traditional ultracentrifugation microvesicle isolation methods, yielding purified extracellular vesicles in just 20 minutes. Using technology developed with Exosome Diagnostics, Inc., the kits use a spin column format and specialized buffers to purify exosomes from prefiltered plasma; up to 4 ml using the maxi kit, and 1 ml using the midi kit. Total RNA is then extracted using QIAGEN miRNeasy technology. The entire procedure, from serum/plasma to RNA, takes only 1 hour.
The exoRNeasy Serum/Plasma Starter Kit provides both midi and maxi exoEasy columns, enabling analysis at varied volumes of prefiltered plasma.
Procedure
The total procedure for isolating RNA from extracellular vesicles comprises two phases: exosome purification and RNA isolation (see figure The exoRNeasy Serum/Plasma Maxi Kit workflow — sample to microvesicles to total RNA in just 1 hour).
In the exosome purification stage, prefiltered plasma (with particles larger than 0.8 μM excluded) is mixed with Buffer XBP and bound to an exoEasy membrane affinity spin column. The bound exosomes are washed with Buffer XWP, and then lysed with QIAzol. In the RNA extraction step, chloroform is added to the QIAzol eluate, and the aqueous phase is recovered and mixed with ethanol. Total RNA, including miRNA, binds to the spin column, where it is washed three times and eluted.
Applications
exoRNeasy Serum/Plasma Kits are highly suited for isolating total RNA, including mRNA and miRNA, from microvesicles found in serum or plasma samples.
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